Mononuclear phagocytes are derived from progenitor cells in the bone marrow which differentiate into blood monocytes and then enter the tissues to occupy specific niches (Hume et al. 2002). They constitute the first line of defense against pathogens, maintain homeostasis, and have trophic functions ranging from bone morphogenesis to neuronal patterning in sexual development, from angiogenesis to adipogenesis (Pollard 2009). Macrophage colony-stimulating factor (CSF1) is required for normal differentiation, proliferation and survival of macrophage lineage cells (Sweet and Flume 2003; Chitu and Stanley 2006; Bonifer and Hume 2008). Mice and rats bearing mutations in the CSF1 gene (i.e. op/op mice and tl/tl rats) show a mononuclear phagocyte deficiency, and their important developmental abnormalities, such as reduced somatic growth, perinatal mortality, osteopetrosis, neurological and reproductive defects, highlight many of the macrophage trophic roles mentioned above (Marks et al. 1992; Pollard 1997, 2009; Ryan et al. 2001).
Although CSF-1 exists in a number of isoforms, most biochemical studies have focused on the minimal biologically active fragment i.e. the 154 N-terminal amino acids, common to all isoforms. In a full-length CSF1 molecule, this receptor-binding region is preceded by a 32-amino-acid signal peptide, and followed by a variable spacer region, a 24-amino-acid transmembrane region and a 35-amino-acid cytoplasmic tail. The tertiary structure of the active fragment of CSF1 forms a short-chain four-helical bundle (A, B, C and D) with small regions of beta-sheet (1 and 2). The helices are paired into A-C and B-D by intrachain disulfide bonds, while one interchain disulfide bond generates a mature homodimer with a two-fold rotation axis (Pandit et al. 1992). The crystal structure of mouse CSF1 bound to its receptor, CSF1R, was recently solved to a resolution of 2.4 Å, showing that CSF1 N-terminal segment (residues 6-15), helix B (residues 55-66) and helix C (residues 79-85) are implicated in receptor binding. (PDB code 3EJJ) (Chen et al. 2008).
All CSF1 effects are mediated through binding to the CSF1 receptor (CSF1R), a glycoprotein of 165 kDa that is encoded by the c-fms proto-oncogene (Dai et al. 2002). CSF1R is a member of the type III protein tyrosine kinase family, along with PDGFRA, PDGFRB and c-kit, and shares with other family members a characteristic extracellular region of five immunoglobulin-like domains (D1 to D5), a single transmembrane helix and a intracellular tyrosine kinase domain (Rosnet and Birnbaum 1993). CSF-1 associates tightly with the receptor (KD=0.4 nM at 37° C.) in a 2:2 stoichiometry (Guilbert and Stanley 1986). This binding involves the CD loop (residues 141-151) and the EF loop (residues 168-173) of D2, as well as the BC and DE loops (residues 231-232 and 250-257 respectively) of D3 (Chen et al. 2008).
The expression of CSF1R on the cell surface is amongst the earliest events in macrophage lineage commitment, and is mostly restricted to these cells throughout embryonic development as well as in adults (Lichanska et al. 1999) Like other myeloid promoters, the proximal CSF1R promoter lacks the classic TATA box and GC-rich sequences but contains recognition sites for AML1 transcription factors, and for transcription factors of the C/EBP and Ets families, including the myeloid-restricted transcription factor PU.1 (Reddy et al. 1994; Himes et al. 2005; Bonifer and Hume 2008). Expression of CSF1R is also controlled by FIRE (Fms Intronic Regulatory Element) a highly-conserved enhancer element in the first intron (Nimes et al. 2001; Sasmono et al. 2003).
Most of the phenotypic defects seen in the op/op mice including reproductive defects and perturbations in organ development, are even more severe in the CSF1R knockout mice (Dai et al. 2002). First attributed to the availability of maternal-derived CSF1, these observations can now be explained by the recent discovery of a second ligand for the human CSF1R, designated IL34, with an activity on monocyte viability. IL34 was purified as a homodimer composed of 241-amino acid monomers, and shown to be expressed mostly in the brain but also in many other tissues including heart, spleen, lung, liver, kidney and thymus (Lin et al. 2008).
WO2008/031172 describes the use of CSF1 to promote organ development in warm-blooded animals and in particular premature human foetuses/embryos. Furthermore, WO03028752 describes methods and compositions for modulating immune responses in animals, said methods comprising modulating CSF1 activity.
The chicken has been used widely in studies of early embryonic myelopoiesis (Lichanska et al. 1999; Lichanska and Hume 2000), but compared to our knowledge of the mammalian mononuclear phagocyte system, our knowledge of avian systems is rather limited and neither WO2008/031172 or WO03028752 describe the existence of an avian CSF1 gene. Indeed, there are only two characterized colony-stimulating factors in chickens. Chicken GM-CSF (CSF2) has been cloned and shown to drive the proliferation of chicken bone marrow cells (Avery et al. 2004). The other described chicken CSF, myelomonocytic growth factor, has recently been shown to be the chicken ortholog of G-CSF (CSF3) (Gibson et al. 2009). Apparent orthologs of the GM-CSF receptor alpha chain, and the beta chain, shared with the IL3 receptor, are annotated in the chicken genome (Ensembl). So far, no function for the CSF1R has been demonstrated in birds, CSF1 was believed to be absent from avian genomes and IL34 had not been recognized (Kaiser 2007).
CSF1 and IL34 in humans were reported to have little obvious homology, and in mammals at least, CSF1 has evolved rather rapidly. The existence of two ligands for a single receptor is difficult to maintain across evolution if they evolve independently of each other and of the receptor.